Selenium is an essential trace element that is incorporated into proteins as selenocysteine. My long-term goal is to understand the mechanism of selenoprotein biosynthesis in mammalian cells. Selenoproteins are synthesized by a novel pathway that involves the co-translational incorporation of selenocysteine at a UGA codon in the mRNA. This mechanism is unusual, since UGA is normally read as a translational stop codon. In eukaryotes, the recognition of UGA as selenocysteine requires the 3' untranslated region (UTR) of the mRNA which contains a stem-loop structure. However, little is known as to how the mammalian ribosome distinguishes a UGA that encodes selenocysteine from a UGA that signals termination. The goal of this proposal is to investigate the mechanism of selenocysteine incorporation, using phospholipid hydroperoxide glutathione peroxidase (PhGPx) as a model. PhGPx is a selenoperoxidase that protects cells from oxidative damage by degrading lethal lipid hydroperoxides in membranes. We recently isolated a full-length cDNA clone that encodes rat PhGPx. Although the PhGPx 3' UTR has the potential to form a stable stem- loop structure, it differs in sequence and structure from other selenoprotein mRNAs. In this proposal, we will test the hypothesis that selenocysteine incorporation involves interactions between sequences in the PhGPx 3' UTR and cellular proteins. (Aim 1) Site-directed mutagenesis and secondary structure analyses will be used to identify the sequences and structures in PhGPx mRNA that are required to decode UGA as selenocysteine. (Aim 2) Gel retardation and UV crosslinking techniques will be used to identify cellular proteins that bind to this RNA sequence to mediate selenocysteine incorporation. The binding protein will be purified to homogeneity by biochemical approaches, including RNA affinity chromatography. To isolate cDNA clones that encode the binding protein, a bacterial expression library will be screened with a 32P-labeled RNA that contains the sequence required for selenocysteine insertion. Alternatively, an oligonucleotide that corresponds to the peptide sequence of the purified protein will be used for cloning by the polymerase chain reaction. (Aim 3) The structure and function of the binding protein will be analyzed to identify the domain involved in RNA-binding. We will also test whether the binding protein is regulated by selenium or oxidative stress. These studies will provide insight into the cellular events that occur in selenium deficiency.